Spot-Trap

Immunoprecipitation of Spot-tagged protein. Input (I), non-bound (FT) and bound (B) fractions were separated by SDS-PAGE followed by Coomassie staining and Western blotting. Western blot indicates high effectiveness of pull-down.

Spot-Trap is a very low background affinity reagent. See benchmark of Spot-Trap Agarose and Magnetic Agarose with other suppliers’ capture tools. Immunoprecipitations were conducted from HEK293T cell lysates without Spot-Tag present and normalized for equal binding capacity. The IPs were done according to respective manufacturers’ protocols. It is apparent that Spot-Trap does not only lack heavy and light chain antibody contaminations but also unspecific binding. This makes Spot-Trap a superior affinity capture tool for single band purification of Spot-tagged proteins.

Elution of affinty purified, Spot-tagged protein. Input (I), non-bound (FT) and eluted (E1, E2 and E3) fractions were separated by SDS-PAGE followed by Coomassie blue staining and Western blotting. Elution with Spot-peptide. Western blot indicates effective elution.

Description

The ChromoTek Spot-Trap^® is an affinity matrix for immunoprecipitation of Spot-tagged proteins.
It consists of an anti-Spot-Tag Nanobody/ V_HH, which is conjugated to beads.

More information about the Spot capture and detection tag can be found here.

Applications
Immunoprecipitation (IP)/ Co-IP
IP/ Co-IP for mass spec (MS) analysis
For protein purification use Spot-Cap

When use Spot-Trap? When use Spot-Cap? Click here.

Specificity
Spot-Tag is an inert 12 amino acid peptide tag with the sequence PDRVRAVSHWSS.

Formats/matrices
Spot-Trap Agarose
Spot-Trap Magnetic Agarose
Spot-Trap Magnetic Beads
Spot VHH, recombinant binding protein
iST Spot-Trap Kit for IP plus sample preparation for MS

Synonyms
anti-Spot V_HH, Spot binding protein, Spot Nanobody, or anti-Spot single domain antibody fragment (sdAb)

Benefit from Spot-Trap for Immunoprecipitation (IP)

Reduced background
High binding affinity, low dissociation constant KD of 6 nM for Spot fusion proteins
Harsh washing conditions
High reproducibility
Short incubation time of 30-60 minutes
Extraordinary low background compared to other affinity resins

Comparison of matrices & properties

We offer Spot-Trap as Agarose, Magnetic Agarose, and Dynabeads.

	Spot-Trap Agarose	Spot-Trap Magnetic Agarose	Spot-Trap Magnetic Beads
Low background	+++	++	++
Binding capacity	+++	+++	+
Size Spot-tagged protein*	Small to large	Small to large	Small to very large
Bead separation	Centrifugation	Magnetic	Magnetic

* Does depend on protein size and shape, protein multimers, complexes and interaction partners

What Spot-Trap shall I use?

Spot-Trap Agarose, when lowest background and high binding capacity IP is needed.
Spot-Trap Magnetic Agarose, when magnetic separation and high binding capacity IP is needed.
Spot-Trap Magnetic Beads, when very large proteins/complexes are investigated, and magnetic separation is needed for IP.
iST Spot-Trap kit for immunoprecipitation plus sample preparation for mass spectrometry (MS)

Specificity

Spot-Trap binds to the sequence motif PDRVRAVSHWSS.

Spot-Trap is a very low background affinity reagent. See benchmark of Spot-Trap Agarose and Magnetic Agarose with other suppliers’ capture tools. Immunoprecipitations were conducted from HEK293T cell lysates without Spot-Tag present and normalized for equal binding capacity. The IPs were done according to respective manufacturers’ protocols. It is apparent that Spot-Trap does not only lack heavy and light chain antibody contaminations but also unspecific binding. This makes Spot-Trap a superior affinity capture tool for single band purification of Spot-tagged proteins.

About Spot

ChromoTek’s proprietary and novel Spot-System is the first peptide-tag specific Nanobody for universal capture & detection applications. It comprises Spot-Tag^®, an inert 12 amino acid peptide-tag (PDRVRAVSHWSS), which has been affinity optimized, and the universal, rugged Spot-Nanobody that specifically binds to Spot-tagged proteins with high affinity.
Spot-Tag peptide is derived from the protein beta-catenin: its sequence has been optimized for higher affinity binding to the Spot-Tag Nanobody. For further information see here.